The Effect of Epigallocatechin Gallate on the Dentin Bond Durability of Two Self-etch Adhesives.

STATEMENT OF THE PROBLEM
Self-etch adhesives can activate matrix metalloproteinase (MMP) which hydrolyzes organic matrix of demineralized dentin. Epigallocatechin gallate (EGCG), especially found in green tea, could inhibit the activation of MMP.


PURPOSE
The aim of this study was to evaluate the effect of adding Epigallocatechin gallate (EGCG) into two types of adhesives on dentin bond strength.


MATERIALS AND METHOD
In this experimental study, 64 extracted third molars were randomly divided into 16 groups. Clearfil SE Bond and Filtek Silorane System with 0 µM, 25µM, 50µM, and 100µM concentration of 95% EGCG were used for bonding. Following the bonding and fabrication of beams (1±0.1 mm(2)) and storage in distilled water, the specimens were subjected to thermal cycles. Microtensile bond strengths of 8 groups were examined after 24 hours and others were tested after 6 months. The fracture modes of specimens were evaluated by stereomicroscope and SEM. Data were analyzed by three-way ANOVA and t-test (α = 0.05).


RESULTS
The results of the three- way ANOVA test showed that types of bonding, storage time and interactive effect of EGCG concentration and bonding influenced the bond strength of specimens significantly (p<0.05). The results of the t-test indicated that storage time only had significant effect on bond strength of Clearfil SE Bond with no EGCG (p= 0.017). The most common failure modes in Filtek Silorane System groups and Clearfil SE Bond groups were adhesive and mixed/cohesive, respectively. The results of SEM at different magnifications showed that most fractures have occurred in the hybrid layer.


CONCLUSION
Although adding 100 µM volume of EGCG to Clearfil SE Bond can preserve the dentin bond, incorporation of EGCG in the silorane system, especially in high concentrations, decreases the bond strength after 6 months.


Introduction
In recent years, development of dentin bonding agents has been considerably accelerated. A widely noted important aspect is to increase the durability of resin adhesives to dentin. Bond degradation occurs through water sorption, [1] hydrolysis of ester linkages of methacrylate resins, [2] or activation of endogenous dentin matrix metalloproteinases (MMPs). [3] MMPs are a group of host-derived proteolytic enzymes which are trapped within mineralized dentin matrix and are able to hydrolyze the organic matrix of demineralized dentin. Odontoblasts produce a large number of MMPs in form of proenzymes. After activation, these proenzymes degrade extracellular matrix components. Unfortunately, self-etch and etch-and-rinse adhesives can activate MMPs. [4] Clinically applied self-etching dental adhesives are acidic (pH= 1.5-2.7) and can activate the MMPs in dentin without denaturing these enzymes during the bonding procedure that results in a 14-to 15-fold increase in collagenolytic activities. [5] Moreover, it has been proved that zoledronate, a third generation bisphosphonate, has the ability to inhibit MMP proteolytic activities. [6][7] Soybean unsaponifiables, avocados, [8] and oleic acid [9] have demonstrated effective MMP-inhibition in vitro. These polyphenols are used in dentistry to prevent loss of alveolar bone in periodontal diseases, to reduce dentin loss in erosion and abrasion, and to prevent caries. The mechanism has been suggested to be due to the inhibitory effect of polyphenol metalloproteinases. [10] Green tea polyphenols, especially epigallocatechin gallate (EGCG), are able to inhibit the activation of proMMP-2, MMP-2 and MMP-9. [11][12] There are not enough data in the literature about the effect of EGCG as an MMP inhibitor on durability of the bond between resin adhesives and dentin. Therefore, the incorporation of EGCG into the adhesives may positively affect dentin bond strength. Thus, the short-term effect of the preservation of dentin bond strength based on adding green tea polyphenol epigallocatechin gallate should be tested. Green tea polyphenol epigallocatechin gallate (EGCG) was added to two types of two-step self-etching adhesives in order to investigate the preservation of dentin micro tensile bond strength (μTBS) in short-term.

Materials and Method
Two types of two-step self-etching primer adhesives, Clearfil SE Bond (Kuraray; Osaka, Japan) and Filtek before and after the specimens were cured. The internal reference employed was aromatic C. . .C (peak at 1608cm−1). The calculation formula of the degree of conversion was as presented below: [14] DC % = (1 − (1638cm_1/1608cm−1) peak area after curing/ (1638cm−1/1608cm−1) peak area before curing) × 10 Sixty four unerupted caries-free third molars were collected after an informed consent was obtained from the patients. These molars were stored in 0.2% thymol solution at 37ºC for less than one month. The occlusal enamel surfaces of the teeth were removed until the diameter of the exposed dentin surfaces was 5 mm.
In order to form a homogenous smear layer on the surface of the occlusal dentin, the teeth were polished with a wet 600-grit silicon carbide paper for 30 seconds.
Filtek Z250 (     whereas in Silorane groups, there were no significant differences between non-stored and stored groups (p> 0.05).

Assessment of the failure mode
In the Filtek Silorane System groups, adhesive failure was the most common fracture mode. In the Clearfil SE Bond groups, however, mixed and cohesive failures were the most common fracture modes (Table 3).

SEM observations
The results of SEM at different magnifications showed mixed fracture mode to be the most common failure mode (Figures 1 and 2). In fact, most fractures occurred in the hybrid layer (Figure 3).

Discussion
The preservation of dentin-adhesive bonds increases the clinical efficiency of bonded restorations. In the process of bonding, however, the simplified etch-and-rinse and self-etch adhesives, reactivate the endogenous collagenolytic and gelatinolytic activities. These can be broken down into unprotected collagen fibrils in presence of water derived from water sorption by hydrophilic adhesives. [3] In this study, we used two types of two-  [14] the results of our study showed that adding EGCG into adhesives had no effect on DC.
The results of this study showed that the bond strength of Clearfil SE Bond was considerably higher than that of the Filtek Silorane System. An explanation for this could be that Clearfil SE Bond contains 10- MDP monomer, and the intense chemical adhesion to tooth tissue may be, to some extent, attributed to that.